Physiological And Morphological Differences And Transcriptome Analysis Between Resistant And Susceptible Cultivars Infected With Sclerospora Graminicola In Foxtail Millet

The downy mildew of foxtail millet is an oomycete disease caused by Sclerospora graminicola.It shows different symptoms during the whole growth stage.It can reduce the yield,which seriously restricts the development of millet industry in China.The current understanding of foxtail millet-S.graminicola interaction is limited.In this study,different cultivars of foxtail millet for downy mildew resistance were screened by inoculation both in field and greenhouse.The transcriptome patterns at different stages of infection in resistant and susceptible cultivars were analyzed by using RNA-seq technology,and the changes of physiological,biochemical and ultrastructure of host were studied.It preliminarily explained the molecular mechanism of resistance to downy mildew,which provided a theoretical basis for molecular breeding of foxtail millet.The main results are as follows:1.The pathogenic conditions and the resistance ability of different foxtail millet cultivars were investigated by mixing oospores and seeds.The inoculation conditions were: the ratio of seed to oospore,10:1;sowing depth,3~5cm;temperature,20°C;and relative humidity(RH),55%.By adjusting humidity to 90%,the " abaxial mould " symptom was observed.Combining the disease resistant analysis results both in field and the greenhouse,Jingu 21 was selected as susceptible cultivar whereas GBS as high resistance cultivar.2.Different conditions such as pretreatment,illumination,temperature,soil immersion solution,seedling tissue extract and root exudate were used to explore the optimal conditions for germination of oospores.It was found that the oospores treated with Na Cl O and cultured in root exudate with alternating light and dark at 20°C could promote their germination.3.The ultrastructural observation of 3,5 and 7 leaf stages in resistant and susceptible cultivars infected by S.graminicola by transmission electron microscopy.At 3-leaf stage,there was no significant difference in different resistant cultivars.At 5-leaf stage,the mesophyll cells of susceptible cultivar began to deform,the inner structure of chloroplasts,the arrangement of thylakoids,and the structure of basal lamellae were destroyed,loose,and disordered respectively.But vesicles appeared,which might be secreted by S.graminicola.Only the number of starch grains were increased in resistant cultivar GBS.At 7-leaf stage,the number of osmiophilic granules increased,chloroplasts disintegrated,cell walls disintegrated and broken,organelles disappeared,and the haustorium and oospores formed in susceptible cultivar.The chloroplast granules were disordered,and the chloroplast membrane structure was not affected in resistant cultivar.It indicated that the disintegrating stage of cell structure in whole process of infection was significantly delayed in the resistant cultivar compared with the susceptible cultivar.4.The cell wall degrading enzymes activities of Cx,PMG and PGTE were all gradually increased at the early stage of infection(3-5 leaf stage)in both resistant and susceptible cultivars.The activities of alpha-glucanase and beta-1,3-glucanase increased rapidly at the late stage of infection(5-7 leaf stage),whereas the activities of beta-G and PG enzymes kept increasing during the whole infection process(3-7 leaf stage).It was speculated that cell wall degrading enzymes play an important role in host and S.graminicola.5.By analyzing the physiological and biochemical changes of different resistant millet cultivars inoculated with pathogen,the results showed that the resistance mechanism between resistant and susceptible cultivars to pathogen were different.Downy mildew infection could reduce morphological and photosynthetic parameters in Jingu21 and GBS.And it could change the defense enzymes activities and the content of osmotic adjustment substances in foxtail millet leaves,but the decline of morphological and photosynthetic parameters was less in GBS.The defense enzymes activities and the content of osmotic adjustment substances were higher in GBS.Therefore,plant height,leaf area,chlorophyll content,net photosynthetic rate and POD,CAT,PAL,soluble sugar and proline content could be used as reference index for the comprehensive evaluation of the foxtail millet downy mildew resistance.6.The transcriptome sequencing analysis of 3,5 and 7-leaf stages inoculated with S.graminicola in resistant and susceptible cultivars showed that there were 852,707 and 687 differentially expressed genes in resistant cultivar and 703,1387 and 1215 differentially expressed genes in susceptible cultivar.Using GO enrichment and KEGG metabolic pathway analysis,it was found that the biosynthesis pathway of phenylalanine,keratin,phellodendrine,and wax were playing important roles in disease resistance regulation.Genes encoding 4-coumaric acid-coenzyme,ligase,peroxidase,beta-glucosidase,cytochrome P450,laccase,chitinase,ABC transporter,phenylalanine ammonia lyase and GDSL esterase were significantly induced in resistant cultivar,whereas those genes were inhibited or less expressed in susceptible cultivar,suggesting that these genes played a key role in the interaction between foxtail millet and S.graminicola.In this study,the inoculation methods of downy mildew in foxtail millet were establisted,and the germination conditions of oospores were optimized.The molecular mechanism of foxtail millet in response to the infection of Sclerospora graminicola were preliminarily revealed from the aspects of histology,physiology,biochemistry and transcription level.The results provided theoretical reference for foxtail millet resistance breeding.